The long-term goal of the Zayzafoon laboratory is to develop novel therapeutic approaches for building and retaining bone mass in humans. The immediate goal of this proposed work is to unequivocally determine the role of the Calcineurin (Cn) and Nuclear Factor of Activated T Cells (NFAT) signaling pathway in regulating bone mass. Bone is a highly dynamic structure that is constantly renewing through a process called remodeling. Cn and NFAT are key promoters of osteoclast differentiation and bone resorption. In contrast, the role of Cn and NFAT in osteoblast differentiation and bone formation is not well defined. We discovered that the pharmacologic inhibition of Cn by low concentrations of Cyclosporin A (CsA) increased osteoblast differentiation in vitro and bone mass in vivo. This increase in osteoblast differentiation was associated with a similar increase in the expression of Fra-2, an AP-1 family member. To determine whether Cn directly impacts osteoblast differentiation, we utilized Cre-mediated recombinase technology to create mice lacking the calcineurin regulatory subunit (Cnb1) specifically in osteoblasts. Our results demonstrate that a block in the Cn/NFAT signaling pathway, when restricted to osteoblasts, leads to an increase in bone mass by directly increasing osteoblast differentiation and indirectly decreasing osteoclastogenesis. Therefore, we hypothesize that the Cn/NFAT signaling pathway negatively regulates osteoblast differentiation and bone mass by regulating the expression and activation of AP-1 family of transcription factors. The Specific Aims of this study are: 1. Determine how the disruption of CN/NFAT signaling in osteoblasts affects bone phenotype. 2. Characterize the roles of Cn/NFAT in osteoblasts growth and differentiation. 3. Determine the molecular mechanisms by which Cn/NFAT regulates bone mass. Previous publications characterize the Cn/NFAT pathway as a positive regulator of bone formation. However, the data from mice with osteoblast-specific knockout of this pathway suggest the opposite - that Cn/NFAT is a negative regulator of bone formation. We are dedicated to definitively resolving this issue because of the therapeutic potential of using inhibitors of Cn/NFAT to anabolically increase bone mass. Our preliminary data provide strong evidence that Cn/NFAT is a negative regulator of bone mass. Completion of these aims will enable us to unequivocally determine the roles of Cn/NFAT signaling in osteoblast differentiation and bone mass. The impact of our work may be far reaching, because it will provide better understanding of the mechanisms by which Cn/NFAT regulates osteoblast differentiation and will have important implications in the development of new anabolic drugs targeting Cn/NFAT pathway to treat osteoporosis and bone loss. PUBLIC HEALTH RELEVANCE: We will unequivocally determine the roles of Cn/NFAT signaling in osteoblasts in regulating bone formation and bone mass. The impact of our work may be far reaching, because it will have important implications in the development of new anabolic drugs targeting Cn/NFAT pathway to treat osteoporosis and bone loss.